Abstract

Screening for tumor suppressor genes in breast cancer revealed multiple truncating mutations of PB1, which encodes the BAF180 subunit of the PBAF chromatin remodeling complex. Mutation was associated with loss of heterozygosity of the wild-type allele. BAF180 complementation of BAF180-mutant tumor cells caused G(1) arrest that was dependent on increased expression of the cyclin/cyclin-dependent kinase inhibitor p21/WAF1/CIP1. Endogenous wild-type BAF180 bound to the p21 promoter and was required for proper p21 expression and G(1) arrest after transforming growth factor-beta and gamma-radiation treatment. BAF180 thus functions on two tumor suppressor signaling pathways as a physiologic mediator of p21 expression. We conclude that BAF180 suppresses tumorigenesis, at least in part, through its ability to regulate p21.

A, Genetic alteration of BAF180 in Bx41. Sequence analysis indicates Bx41 has duplication of exon 5, 6 and 7, producing stop codon at codon 222. The stop codon is underlined. B, Genetic alteration of BAF180 in breast cancer and its derived cell line SUM1315. On left, the RT-PCR product of SUM1315 showed an aberrant transcript. The set 2 primers were used. On right, Southern blot of tumor genomic DNA shows duplication of exons in tumor biopsy taken from lymph node (lane 3) and in the cell line from the same patient SUM1315 (lane 4). Rearranged fragment was detected in lane 3 and 4, but was not in lane 1 (unrelated normal blood DNA) and lane 2 (normal blood DNA from the same patient). DNAs were digested with BglII. The RT-PCR product of exon 15 through 17 was used for a probe. C, Genetic alteration of BAF180 in case 337. Exon sequencing revealed a nonsense mutation on exon 18 in a primary tumor sample (case 337). A corresponding normal sequence (case 336) is shown for comparison. D, Domains and mutations of BAF180. The BAF180 cDNA encodes a protein 1635 amino acids. Mutations are indicated with arrows. Homozygous deletion indicated with black bar.

A. Chromatin-IP of BAF180 on p21 promoter. Immunoprecipitation of BAF180 crosslinked to chromatin detects BAF180 binding to p21 promoter with serum from rabbits immunized with BAF180 but not pre-immune serum. No BAF180 binding is detected 7–15 kb outside of the promoter region. B. BAF180 knockdown decreases the level of p21 mRNA. RNAs from siRNA transfected MCF10A cells were subjected to reverse transcription. Quantitative PCR was performed using RT products as the templates and p21 and tubulin primers. The p21 results were normalized to tubulin and quantified with MxPro from Stratagene. C. BAF180 knockdown decreases the level of p21 protein. MCF10A cells were transfected with siRNA using nucleofector (Amaxa) and then lysed on the next day. c, non-targeting oligo as a control; 1, oligo from Dharmacon D-008692-01; 2, oligo from Dharmacon D-008692-02; 3, oligo from Dharmacon D-008692-03.

A. BAF180 knockdown causes cell population shift. MCF10A cells were transfected with BAF180 or non-targeting siRNA and collected 24 hours later. B. MCF10A cells transfected with BAF180 siRNA (B) or non-targeting siRNA (N) BAF180 were treated with 100 pM TGFβ (+) for 24 hours and analyzed by cytometric and western blotting. C. MCF10A cells transfected with BAF180 siRNA (B) or non-targeting siRNA (N) were γ-irradiated and subjected to cytometric and western analysis. The cell cycle arrest and p21 induction elicited by either TGF-β or γ-radiation is markedly attenuated by knockdown of BAF180 due to RNAi.